Cardiac excitation-contraction (E-C) coupling refers to a cascade of Ca²⁺-mediated events whereby membrane depolarization leads to cell contraction. At the subcellular level, E-C coupling occurs within a microdomain of the cardiomyocyte, termed the cardiac dyad. In various forms of heart disease, such as pathological hypertrophy and heart failure, the E-C coupling process is abnormal, in part because of ultrastructural remodeling. Abnormal Ca²⁺ homeostasis (as a result of failed E-C coupling) triggers maladaptive remodeling at the transcriptional level, contributing to pathological myocardial remodeling, hypertrophy, and heart failure. However, it remains unclear whether cardiomyocytes possess a self-protective or homeostatic mechanism that mitigates adverse myocardial remodeling.

Junctophilin-2 (JP2) is a structural protein that organizes the E-C coupling ultrastructural machinery in cardiomyocytes. We previously showed that calpain-mediated proteolytic cleavage of JP2 is key to its down-regulation in the diseased heart after cardiac stress. This cleavage contributes to loss of ultrastructural integrity at cardiac dyads, E-C uncoupling, and dysfunction of Ca²⁺ handling that results in heart failure. Computational analyses predicted that JP2 contains a nuclear localization signal (NLS), as well as an alanine-rich region (ARR) with characteristics of a helix-turn-helix structure, a DNA binding motif. We tested the hypothesis that JP2 encodes a stress-adaptive transcriptional regulator, which transduces mechanical information (E-C uncoupling) into transcriptional reprogramming in the myocardium in the setting of cardiac stress.

Biochemical, mutagenesis, and confocal imaging analyses revealed that stress-induced proteolysis of JP2 liberated an N-terminal fragment (JP2NT) that was imported into the nucleus through its NLS. Further biochemical and microarray assays showed that in the nucleus, JP2NT associated with chromatin and regulated transcription of a wide spectrum of genes via an evolutionarily conserved ARR located in the α-helix region of JP2. Chromatin immunoprecipitation sequencing (ChIP-seq) of JP2NT-overexpressing hearts revealed that it bound preferentially to the transcription start sites (TSSs) of genes, and gel shift studies defined the DNA binding motifs of JP2NT as the TATA box and a MEF2-response element (MRE). Elevation of JP2NT levels by JP2NT overexpression altered the in vivo genomic binding profile of TATA-box binding protein (TBP) and MEF2C. In addition, JP2NT suppressed the transcriptional activity of MEF2C by competing for MRE. Overexpression of JP2NT in mice led to reprogramming of the transcriptome in the setting of stress and attenuated hypertrophic remodeling and the progression of heart failure. Loss of JP2NT function by deletion of the JP2 NLS in mice accelerated the development of hypertrophy and heart failure after cardiac stress.

Our data reveal that calpain-mediated cleavage of JP2 transforms this E-C coupling structural protein into a transcriptional regulator that is shuttled into the nucleus and binds to promoters of target genes, inducing cardioprotective transcriptional reprogramming. These data reveal that cardiomyocytes possess a self-protective mechanism that counters pathological transcriptional remodeling after cardiac stress. Our findings also identify an intrinsic direct connection between ultrastructural remodeling and transcriptional reprogramming in the stressed heart.

Left: E-C …